HIV-1 has a high mutation rate, which contributes to its ability to evade the host immune system, limits the efficacy of antiretroviral drugs and drives the emergence of drug resistance. Drug resistance conferring mutations as well as other viral mutations are primarily attributed to the error-prone nature of reverse transcriptase (RT). An intentional increase in RT-mediated mutations decreases virus infectivity by increasing the mutation rate to a level that is not able to maintain survival of the virus population. The potency by which HIV-1 infectivity can be decreased by increasing RT-mediated errors has led to an initiative to discover small molecules that may increase the HIV-1 mutation rate. An interdisciplinary collaborative team has been assembled to 1) conduct discovery studies to identify new small molecules that increase RT-mediated errors, 2) use molecular analyses to identify the mechanism(s) by which small molecules increase the HIV mutation rate and result in virus extinction, and 3) to assess the mechanism of RT-mediated mutation using biochemical methods. Through preliminary studies, we have identified four small molecules that increase RT-mediated mutations. In order to elucidate the structure-activity relationship driving this increase and to optimize this activity, we will first pursue discovery studies to identify small molecules that can increase RT-mediated errors. The antiviral and mutagenic activities of these molecules will be assessed in cell culture. Second, we will examine the mechanism by which small molecules induce mutations and cause virus extinction in HIV-1 using cell culture methodologies. Here we will examine small molecules that we have already discovered as well as any lead molecules that we identify. Third, we will investigate the mechanism of action using biochemical methods to elucidate the mechanistic basis for increased RT-mediated mutation. Successful completion of these studies will provide deeper insight into the mechanisms of RT-mediated viral mutagenesis and its impact on viral replication and extinction.